Evaluation of thyroid function in patients with thyroid hormone autoantibodies

Evaluation of thyroid function in patients with thyroid hormone autoantibodies

ELSF%II:R S~,IINTII l( PUBI I~1tl R~ I r l t -%%1) Ciinica Chimica Acta 219 (1993) 23-34 Evaluation of thyroid function in patients with thyroid hor...

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ELSF%II:R S~,IINTII l( PUBI I~1tl R~ I r l t -%%1)

Ciinica Chimica Acta 219 (1993) 23-34

Evaluation of thyroid function in patients with thyroid hormone autoantibodies Shigeki Sakata *a, Takashi Komaki a, Toru Ogawa a, Hiroshi Takuno a, Ikuo Matsui a, Hiroshi Sarui a, Noriko Kojima a, Junta Takamatsub, Kiyoshi Miuraa "The Third Department of Internal Medicine, G~fu UniversiO, School of Medicine, Gifu 500. Japan bThe First Department of Internal Medicine, Osaka Medical College, Takatsuki .~69, Japan

(Received 5 October 1992, revi~ion received 29 March 1993; accepted 29 March 1993)

Absh'act

Serum free thyroxine (FT4) levels were measured in patients with Hashimoto's thyroiditis and Graves' disease who were seropositive for thyroid hormone autoantibodies. The methods used were equilibrium dialysis/radioimmunoassay (FT, by equilibrium dialysis, Nichols Institute) and FT4 analog radioimmunoassay (Amerlex MFT4) before and after treatment of sera with 1,7..5% polyethylene glycol (PEG), Furthermore, FT4 measurement in the PEGtreated sera was done using two other analog radioimmunoassays (DPC FT4 kit and N-FT4 Coming). Serum thyrotropin (TSH) concentration before and after i.v, infusion of thyrotropin releasing hormone (TRH) was measured in five cases of Ha~himoto's thyroiditis and four cases of Graves' disease with thyroid hormone autoantibodies, Although FT4 determination by analog tracer radioimmunoassays showed unusually high values for the hormone, results obtained by an equilibrium dialysis/radioimmunoassay method showed compatible values with basal TSH. Furthermore, FT4 concentrations measured with Amerlex MFT4 after treatment of sera with 12.5% PEG, correlated well with the values obtained by equilibrium dialysis/radioimmunoassay (r = 0.98, P < 0.001). Similar results were obtained with two other analog FT4 radioimmunoassays after treatment of sera with PEG. These results indicate that real FT4 values in patients with thyroid hormone autoantibodies are compatible with basal TSH concentrations. The presence of thyroid hormone autoantibodies per se does not affect the control mechanism of the hypothalamo.pituitary-thyroid axis. It was concluded that measurement of basal TSH as well as FT4 values by either equilibrium dialysis/radioimmunoassay or analog radioimmunoassays after PEG treatment pro~,ides clinicians with valid information for assessment of the precise status of thyroid function in patients with thyroid hormone autoantibodies. * Corresponding author. 0009-8981/93/$06.00 © 1993 Elsevier Science Publishers B.V. All rights reserved. SSD1 0009-8981(93)05591-2

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S. Sakata et al. /Ciin. Chim. Acta 219 (1993) 23-34

Key words: Thyroid hormone autoantibodies; Free "1"4;Free T3; Serum thyrotropin; Equilibrium dialysis; Graves' disease; Hashimoto's thyroiditis

1. Introduction

In 1956 Robbins et al. [I] reported the initial case of [13ml]thyroxine (1"4)binding to serum ~-globulin in a patient with papillary carcinoma who had been treated with 1311. By 1986 approximately 120 cases of thyroid hormone autoantibodies had been reported in patients with both thyroidal and non-thyroidal disorders [2-4]. In such cases serum thyroid hormone autoantibodies have been demonstrated to interfere with radioimmunoassay values of serum total and free thyroid hormone concentrations, resulting in a discrepancy between the plasma hormone levels and clinical features [2-4]. As a result, patients may receive unnecessary treatment if the physicians are unaware of the presence of the autoantibodies. Although it is generally accepted that measurement of serum thyrotropin (TSH) serves as the most sensitive and specific indicator of the biologic effects ofcirculating thyroid hormones [5], serum FT4 measurement is preferred in certain pathological conditions [5,6]. It has been shown that these two tests provide complementary information regarding the normality of thyroid gland T4 production and biological action [5]. Thus, precise evaluation of serum thyroid hormone levels together with TSH measurement is recommended in patients with thyroid hormone autoantibodles. Recently, an equilibrium dialysis/radioimmunoassay kit for the measurement of FT4 became available [7]. Using this technique, we measured the FT4 concentration in thyroid hormone autoantibody-positive patients who had Hashimoto's thyroiditis and Graves' disease and compared the values with those obtained with the Amerlex MFT4 method before and after polyethylene glycol (PEG) treatment [8]. To validate the efficacy of PEG treatment, we also measured the PEG-treated sera from thyroid hormone autoantibody.positive patients with other commercially available analog radioimmunoassays. FT4 values thus obtained by various methods were compared with basal TSH values as well as with the patients' clinical findings. 2. Materials and methods

2.1. Thyroid hormone autoantibody.positive cases avd measurement of serum FT4. FT3 and TSH Sera obtained from 17 cases (ten cases of Hashimoto's thyroiditis and seven cases of Graves' disease) with anti-T4 and/or anti-T3 autoantibodies were used for the present investigation (Table I). As controls, serum concentrations of FT4 and TSH were measured in 38 thyroidal disorders without thyroid hormone autoantibodies. The diagnosis of Graves' disease was based on clinical and laboratory evidence such as the presence of hyperthyroidism with suppressed TSH levels, ophthalmopathy, palpable soft and diffusely enlarged thyroid gland and high thyroidal 1231-uptake. Hashimoto's thyroiditis was diagnosed by the presence of rubbery hard enlarged

13/1: 10/F 64/F 22/F 15/!: 40/1: 35/F 25/i: 36/[: 26/1: 32/F 45/F 16/F 49/F 46/F 57/F 31/F

1. Ma.T. 2. Mi.T. 3. K.K. 4. J.O. 5. T.A 6. K.G. 7. E.H. 8. M.M. 9. Y.M. 10. M.I. I !. K.Y. 12. M.O. 13. M.F. 14. H.I. 15. T.B. 16. M.C. 17. M.A.

T3,T4 T3,T4 T4 T4 T3,T4 T3,T4 T3,T4 T3,T4 T3 T3 T3,T4 T3 T3 1"4 T4 T4 T3

THAA

1602 3202 3202 160 2 802 802 202 802 1602 202 202 (--) 102 3202 1602 202 802

TGHA 3202 80" 3202 (--) 202 402 202 802 802 202 802 25602 402 3202 ~--) 202 202

MCHA Hashimoto Hashimoto Hasbimoto Hashimoto Hashimoto Hashimoto Hashimoto Hashimoto Hashimoto Hashimoto Graves" Graves" Graves" Graves" Graves" Graves" Graves"

Diagnosis Hypothyroid Hypothyroid H)~thyroid Hypothyroid Euthyroid Euthyroid Euthyroid Euthyroid Euthyroid Euthyroid Hyperthyroid Euthyroid Euthyroid Euthyroid Euthyroid Euthyroid Euthyroid

Thyroid state 235 240 240 23.0 I 1.8 7.5 4.3 12.0 2.4 2.5 <0.1 0.4 2.7 4.3 0.8 0.8 !.8

TSH n.d. n.d. <3.21 8. ! 0 9.90 <3.21 10.04 1i.20 16.09 n.d. 58.30 14.16 n.d. I i .20 8.75 12.10 5.15

D-FT 4 135.3 ! 3.5 56.1 30.0 325.6 4.37 13.90 12.48 18.79 10.3 68.46 17.89 60.49 32.82 52.25 30.63 22.52

A-FT 4 0.00 I. ! 6 i.16 9.40 9.01 0.51 8.88 10.3 12.87 n.d. 43.37 ! !.84 20.46 7.85 7.59 10.30 8.75

P-FT4 1.63 16.57 0.75 6.16 I. ! 8 8.60 12.32 6.91 3.69 ! 5.2 24.12 5.53 i 7.5 ! 3.69 7.62 4.61 16.91

810 620 32 n.d. 49 !.6 n.d. 12.0 n.d. 6. i !.7 < 0.2 i .8 2.0 n.d. n.d. n.d.

A-FT 3 B-TSH a

i,700 (30') 2,800 (60') 210 (60') n.d. 350 (30') 1!.6 (30') n.d. i 10.0 (30') n.d. 29. ! (60') 5.6 (30') < 0.2 15.9 (30') 20.8 (30') n.d. n.d. n.d.

P-TSH a

TGHA and MCHA represent antithyrogiobulin and antimicrosomal autoantibodieso respectively, measured by a hemagglutination technique. Titer of TGHA or MCHA was expressed by the final dilution of sera tested. Concentrations ofTSH, FT 4 and FT 3 are expressed as mUll, pmoL/i and pmol/l, respectively. D-FT 4, A-FT 4 and P-FT 4 represent FT 4 values obtained by equilibrium dialysisfRIA, Amerlex FT 4 and Amerlex FT 4 after PEG treatment. A-FT 3 indicate Amerlex FT 3. Normal range of each RIA is shown in text. n.d., not determined. aTSH concentrations before (basal TSH, B-TSH) and after (peak value, P-TSH) 500 pg of TRH administration. Parentheses indicate peak time.

Age/sex

Cases

Table 1 Summary of cases of 16 patients with antithyroid hormone autoantibodies

~

.~" ,~:~

26

S. Sakata et ai. /Clin. Chim. Acta 219 (1993) 23-34

goiter and high titers of antithyroglobulin and/or antimicrosomal autoantibodies measured by a hemagglutination technique (TGHA and MCHA, Fuji rebio, Tokyo) [9]. The diagnosis of Hashimoto's thyroiditis was confirmed by ultrasonography of the thyroid gland which shows enlargement with low and non-homogeneous echo density. The presence of anti-T4 and/or anti-T3 antibodies was confirmed by the previously reported method which use acidified dextran-coated charcoal prior to the removal of any endogenous and/or exogenous thyroid hormones [2,10]. Serum concentration of FT 4 was measured by an analog radioimmunoassay kit (A-FT4, Amerlex MFT4, Amersham International, Tokyo; normal range 10.0-27.2 pmol/I) and equilibrium dialysis/radioimmunoassay kit (D-FT4, FT4 by equilibrium dialysis, Nichols Institute, San Juan Capistrano, CA; normal range 10.2-23.2 pmol/i). Serum concentration of FT4 was also measured by a modification of the analog radioimmunoassay kit (P-FT4). Briefly, an equal amount of 25% PEG (PEG 6,000, Wako Pure Chemicals, Osaka) which had been dissolved in phosphate-buffered saline (PBS, pH 7.4) was added to patients' sera, followed by mixing and centrifugation (Kubota KM-15200, 10,000 rev./min for 10 min at 4°C) [8]. FT4 concentration in the supernatant was measured by an analog radioimmunoassay (Amerlex MFT4). In order to examine the validity of this method, FT4 measurement of thyroid hormone autoantibody-positive sera from a case of Graves' disease and three cases of Hashimoto's thyroiditis {Table 2) before and after PEG treatment was done using two other commercially available radioimmunoassay kits (DPC FT4 kit, Diagnostic Products Corporation, Los Angeles, CA, normal range 9.0-27.2 pmol/I; N-FT 4 Corning, Chiba-Corning, Tokyo, normal range 13.5-35.4 pmol/I). Serum concentration of FT.~ was measured by a commercially available analog radioimmunoassay (A-FTa, Ameriex MFT.~, normal range 4.2-9.1 pmol/I). Serum concentration of TSH was measured by an immunoradiometric assay (Spack TStl, Daiichi Isotope Lab, Tokyo, normal range 0.2-5.0 mU/I). Analog tracers used by radioimmunoassay kits were as follows: Amerlex MFT4, [t:Sl]polyaminocarboxy T4, |)PC FT4, [l:Sl]succinic acid amide "!"4,N-FT4 Coruing, [l:~l]imidazol¢ acetamide T4.

2.2. Measurement of TSH heJbre and after ttt.;,rotropin releasing ht)nntme administration The thyrotropin releasing hormone (TRlt) infusion t¢st was done in seven (cases I, 2, 3, 5, 6, 8 and 10) and four cases {cases I I, 12, 13 and 14), respectively, with thyroid hormone autoantibody-positive Hashimoto's thyroiditis and Graves' disease by i.v. injection of 500 ~g of TRH (Tanabe Pharmaceutical Co., Osaka) {Table I), As a control, the same test was done in 27 thyroid hormone autoantibody-negative cases with thyroidal disorders. Serum concentrations of TSlf before and 30, 60, 90 and 120 rain after i.v. injection of TRH ~ere measured. Because measurement of TSH concentration after 90 rain TRH infusion was lacking in two thyroid hormone autoantibody-positive cases, response of TSH after TRH administration was expressed by the highest TSH values (peak TSH) which implies a peak value after "I'RH administration. It was also expressed by integrated TSH values (E TSH) which imply a sum of basal, 30, 60 and 120 min TSH values. Two thyroid hormone autoantibodypositive hypothyroid patients with Hashimoto's thyroiditis {cases I and 2, Table I) showed extremely high basal, peak and sum of TSH values {TSH values before and

Hashimoto Hashimoto Hashimoto Hashimoto Graves"

3. K.K. 4. J.O. 4. J.O. 6. K.G. 13. H.I.

51.0 260.0 5.4 95.9 0.7

TSH

<3.2 <3.2 15.4 < 3.2 23.9

D-FT 4

75.2 48.4 43.2 24.6 38.1

A-FT 4

2.6 3.5 14.2 6.4 18.0

A-FT4 a

37.5 15.4 28.4 29.9 99.1

DPC-FT 4

3.9 2.6 15.6 4.0 17.8

DPC-Fr4 a

9.3 14.9 31.8 19.8 38.6

Corning-FT4

3.2 2.8 18.2 2.6 18.7

Corning-FT4 a

Concentrations of TSH and FT 4 are expressed as mU/I and pmol/i, respectively. The case numbers are the same as those in Table I. In case J.O., ['1"4 results in two sera obtained before and after "1"4treatment are shown. aValues after treatment of sera with 12.5% PEG.

Diagnosis

Case

Table 2 F'I" 4 values measured by equilibrium dialysis/RIA (D-FT41 and analog RIAs

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.c,u

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S. Sakata et al. /Ciin. Chun. Acta 219 (1993) 23-34

30, 60 and 120 min after TRH administration in case I were 810, !,700, 1,600 and 1,050 mU/I and those in case 2 were 620, 1, 100, 2,700 and 2,800 mU~, which implies that basal, peak and sum of TSH values of case I were 810. 1,700 and 5,160 mU/l, respectively and those in case 2 were 620, 2,800 and 7.220 muff, respectively). A hyperthyroid patient with Graves' disease (case 12, Table I) showed undetectable TSH values (< 0.2 mU/l) before and after TRH administration. The results obtained from these three cases were not included for the comparison with those of control subiects (see Fig. 3(B)). Basal, peak and [: TSH in control subjects ranged from 0.4 to 42.1.5.8 to 31.2 and 13.8 to 750 mU/l, respectively and those in six thyroid hormone autoantibody-positive patients ranged from 1.6 to 49.0, 5.6 to 350 and 15.0 to 849 mU/i, respectively. Since it is known that TSH values show a normal distribution after log transformation [ I 1,12] statistical analyses were done after log transformation of TSH values. Statistical analyses were done according to the paired or unpaired Student's t-test. Correlation between log TSH and FT4, D-FT4, or P-FT4 was made by linear regression analysis and Spearman's correlation coefficient by ranks. Differences were considered to be significant if P < 0.05. 3. R e s u l t s

3.1. Correlation between serum concentration of FT~ and FSH A significant inverse correlation was observed between log TSH and A - [ " T 4 values in untreated primary hypothyroid, euthyroid and hyperthyroid subjects without thyroid hormone autoantibodies by both linear regression analysis (r = -0.81, P < 0.01) and Spearman's rank analysis (r = -0.73, P < 0.01) (Fig. I(A)). Such significant inverse correlation was not noted between A-FI'4 and log TSH values in thyroid hormone autoantibody-positive cases in both statistical analyses (data not shown). Correlation between D-FT.~ or P-I:T4 and log "FSH in patients with thyroid hormone autoantibodies is shown in the middle (B) and lower panel (C) of Fig. I, respectively. There was a significant inver,~ correlation between log TSH and D-FT~ (linear regression analysis: r---0.63, P < 0.01~ Spearman's rank analysis: r = -0.60, P < 0.01) or P-FT4 (linear regression analysis: r = -0,70, P < 0.01~ Spearman's rank analysis: r -- -0.66, P < 0.01)) values, in addition, there was a striking significant positive correlation between values of D-FT4 and P-FT4 (linear regression analysis: r = 0.98, P < 0.01: Spearman's rank analysis: r = 0.97, P < 0.01)(Fig. 2). .~.2. Serum TSH concentration beJi~re and after TRH administration There was a significant correlation between basal TSH concentration and peak values (linear regression analysis: r = 0 . 9 7 , P < 0.01, Fig. 3(A)) or E TSH values (linear regression analysis: r = 0.97, P < 0.01, data not shown) in thyroid hormone autoantibody-negative patients with thyroidal disorders. This was also true in eight thyroid hormone autoantibody-positive patients. There was a significant positive correlation between basal and peak TSH values (linear regression analysis: r =: 0.99, P < 0.01; Spearman's rank analysis: r -- 0.97, P < 0.01) ~Fig. 3(B)) or ~ TSH values (linear regression analysis: r = 0.99, P < 0.01, data not shown) in thyroid h~xmone autoantibody-positive patients.

29

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Fig, I, Correlation between log TSH and FT4 concentration by an analog FT4 radioimmunoassay (Amerlex MFTa) in patients with thyroid disorders without thyroid hormone autoantibodies (A), an equilibrium dialysis/radioimmunoassay (FT4 by equilibrium dialysis) in patients with thyroid hormone autoantibodies (B), and an analog FT4 radioimmunoassay (Amerlex MFT4) after polyethylene glycol treatment of thyroid hormone autoantibody-positive sera (C). More than one serum per patient with thyroid hormone autoantibodies were tested. The middle solid line shows the linear regression line of best fit and the two external dotted lines indicate 95% confidence intervals.

3.3. FT4 measurement o f sera after PEG treatment A-FT4 concentrations after PEG treatment of thyroid hormone autoantibodynegative sera with hyperthyroid, euthyroid, and hypothyroid subjects are 90.5 + 4.7%, 98.1 ± 8.1 and 105.4 ± 5.5%, respectively, of values witbr"Ut the

S. Sakata et aLI C/in. Chim. Acta 219 (1993) 23-34

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Fig. 2. Correlation between the FT4 value obtained by FT 4 equilibrium dialysis (D-FT 4) and Amerlex MFT 4 after polyethylene glycol treatment (P-FT4) of sera in patients with thyroid hormone autoantibodies. The middle solid line shows the linear regression line of best fit and the two external dotted lines indicate 9 5 % confidence intervals,

treatment. Although A-FT4 concentration in hyperthyroid patients declined significantly after PEG treatment (paired t-test, P < 0.01), the magnitude of decrease stayed in the range of less than 10%. in euthyroid and hypothyroid subjects, no significant difference was obtained in FT4 values before and after PEG treatment. A-FT4 concentrations in sera from patients with Hashimoto's thyroiditis or Graves' disease with thyroid hormone autoantibodies before and after PEG treatment are shown in Fig. 4. FT4 values after PEG treatment were consistent with those of basal TSH. FT4 values of five thyroid hormone autoantibody-positive sera before and after PEG treatment by three different commercially available analog radioimmunoassays are shown in Table 2. Although FT4 values gave spuriously high values in all the patients, values after PEG treatment were compatible with patients' clinical findings and basal TSH values. 4. Discussion

Almost all patients with thyroid hormone autoantibodies that have been presented had discrepancies between physical findings and results of radioimmunoassays for free and/or total T3 and "!"4[2-4]. This problem was caused by the in vitro interference of thyroid hormone autoantibodies in the radioimmunoassays for total or

31

S. Sakata et aL / Clin. Chim, Acta 219 (1993) 23-34

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Fig, 3. Correlation between basal and peak TSH values obtained from sera of patients with thyroidal disorders without (upper panel, (A)) or with thyroid hormone autoantibodies (lower panel, (B)), The middle solid line shows the linear regression line of best fit and the two external dotted lines indicate 95% confidence intervals.

free T~ or "!"4by competing with the binding of radiolabeled thyroid hormones or their analog tracers by antithyroid hormone antibodies raised in animals [2]. This will therefore lead to difficulty in evaluating true thyroidal status in patients with thyroid hormone autoantibodies [13]. The prevalence of thyroid hormone autoantibodies which interfere with thyroid hormone radioimmunoassays could be rare, being 1 out of 2,460 sera from thyroidal disorders in one report [14]; however, another report describes the prevalence of thyroid hormone autoantibodies with or without interference of the radioimmunoassay in Hashimoto's thyroiditis as being as high as 40% [15]. It has been shown that measurement of TSH serves as the most sensitive and specific indicator of thyroid hormone action [5]. However, serum TSH as an indicator of thyroid hormone action in patients with thyroid hormone autoantibodies has not been established. In addition, interference from increased nonspecific binding [16], human antibody to bovine TSH [17,18], anti-mouse igG [19], paraproteins [20] and an unidentified substance [21] have also been reported for TSH radioimmunoassays. Furthermore, a case of anti-T3 antibodies associated with

32

S. Sakata et al. I Clin. Chim. Acta 219 (1993) 23-34 ! t

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Fig, 4. Amcrlex MFT.s concentration in sera from patients with Hashimoto's thyroiditis (0.0) and Graves' disease (A,&) before (open circle and triangle) and alter (closed circle and triangle) polyethylene glycol treatment of sera obtained from thyroid hormone autoantibody-positivc patients. Shaded area indicates normal range.

TRH deficiency [22] has been reported, It has been shown that serum F'r4 gives not only complementary information on TSit but is also a cost.effective thyroid screeqing test in certain pathological conditions [5,6]. Although the above-mentioned interference of TSH radioimmunoassays could clinically be a rare condition, it is important to evaluate not only basal TSH but also real serum free thyroid hormone concentrations in patients with thyroid hormone autoantibodies who have discordant radioimmunoassay results of both tree thyroid hormone and TSH with physical findings. Recent availability of all equilibrium dialysis/radioimmunoassay kit for FT4 measurement which uses dialysis cells with which undiluted serum samples can be dialyzed, followed by measurement of dialysate T4 by a sensitive radi0im munoassay [7], enabled us to measure serum FT4 concentration in thyroid hormone autoantibody-positive patients. There was a significant inverse correlation between serum TSH and FT4 values obtained from equilibrium dialysis/radioimmunoassay kit in patients with thyroid hormone autoantibodies. We have previously reported that FT3 concentration after 12.5% PEG treatment gives compatible values with serum TSH concentration and patients' thyroid state [8]. The clinical usefulness of this method was confirmed by studies from another laboratory [23]. it was shown that FT4 values by this method (P-FTa have a strik-

s. Sakata el al./ Ciin. Chim. Acta 219 (1993) 23-34

33

ing significant positive correlation (r = 0.98) with those obtained by equilibrium dialysis/radioimmunoassay. In addition, in patients with thyroid hormone autoantibodies, serum FT4 concentration by three different analog radioimmunoassays after PEG treatment showed compatible values not only with TSH concentration but also with FT4 values obtained by equilibrium dialysis/radioimmunoassay. Thus, in places where equilibrium dialysis/radioimmunoassay is not available, treatment of sera with PEG, followed by radioimmunoassay is valid in the precise evaluation of F T 4 concentration in patients with thyroid hormone autoantibodies. Despite the possibility that thyroid hormone autoantibodies could enter into the anterior pituitary thyrotroph, resulting in an abnormal hypothalamo-pituitarythyroid axis in patients with thyroid hormone autoantibodies, their thyroidal states were compatible with serum concentrations of TSH as well as FT4 values obtained from equilibrium dialysis/radioimmunoassay and analog radioimmunoassays after PEG treatment. In addition, the response of TSH after TRH infusion was similar between thyroid hormone autoantibody-negative and -positive patients with thyroidal disorders. From these results, it was concluded that the presence of thyroid hormone autoantibodies per se does not affect the hypothalamo-pituitary-thyroid axis in patients with the autoantibodies. Thus, measurement of not only basal TSH but also FT4 values by either equilibrium dialysis/radioimmunoassay or analog tracer radioimmunoassays after PEG treatment of sera is recommended for precise evaluation of the real thyroid state in patients with thyroid hormone autoantibodies.

S.

Acknowledgments

We appreciate the help of Nihon Medi & Physics Co. (Nishinomiya) in the measurement of the FT4 of patients' sera with thyroid hormone autoantibodies by the equilibrium dialysis/radioimmunoassay kit. We thank DPC Japan (Tokyo) and Chiba-Corning (Tokyo) for radioimmunoassay kits. The work was supported by a grant for scientific research from the Ministry of Education, Science and Culture of Japan (Grant no. 04671467).

6. References ! RobbinsJ, Rail JE, Rawson RW. An unusual instanceof thyroxinebindingby human serum ~amma globulin. J Clin Endocrinol Metab 1956:18:573-579. 2 Sakata S, Nakamura S, Miura K. Autoantibodies against thyroid hormones or iodothyronine. Implications in diagnosis, thyroid function, treatment, and pathogenesis. Ann Intern Mcd 1985;103:579-589. 3 BenvengaS, Trimarchi F, Robbins J. Circulatingthyroid hormone autoantibodies. J Endocrinol Invest 1987;10:605-619. 4 PremachandraBN, Blumenthal HT. Significanceof thyroid hormone autoantibodies. In: Walfish PG, Wall JR, Volpe R, eds. Autoimmunity and the thyroid. New York: Academic Press, 1985:189-215. 5 Nicoloff,IT,Spencer CA. The use and misuse of the sensitivethyrotropin assays. J Clin Endocrinol Metab 1990;71:553-558. 6 SpencerC, Eigen A, Shen Det al. Specificityof sensitive assay of thyrotropin (TSH) used to screen for thyroid disease in hospitalized patients. Clin Chem 1987:33:1391-1396.

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